JP6934972B2 - Screening method for peroxiredoxin expression enhancer - Google Patents

Description

The present invention relates to a peroxiredoxin expression enhancer.

Peroxiredoxin is known as an enzyme involved in antioxidant activity, which plays an important role in maintaining biological functions because it prevents denaturation and damage of proteins, lipids, and nucleic acids by its antioxidant activity.
In humans, the existence of six isoforms of peroxiredoxin is known. Among them, peroxiredoxin 1-3 is present in the epidermis, expression of peroxiredoxin 1-2 increases as it is closer to the granular layer, and peroxiredoxin 3 is more expressed in the basal layer.

Further, peroxiredoxin is known to suppress active oxygen generated by UV, UVA is known to increase the expression of peroxiredoxin 1, and UVB is known to increase the expression of peroxiredoxin 2.
In addition, it has been suggested that increased expression of peroxiredoxin is involved in prolonging the life span, and in particular, decreased expression of peroxiredoxin 6 is involved in increasing the incidence of skin cancer. (See, for example, Non-Patent Document 1).

Dried fruits of jujube (Ziziphus jujuba), which is a plant belonging to the genus Jujube of the family Buckthorn (Rhamnaceae), or related species, are also called taiso and are used as Chinese herbs. In recent years, the extract has been incorporated into an external preparation for skin as having a predetermined function.
For example, Patent Document 1 describes a melanin-containing keratinocyte division-promoting agent containing an extract of jujube fruit and the like, and a pigmentation inhibitor containing the same. Further, Patent Document 2 describes that a skin cosmetic containing a taiso extract has an anti-aging effect and a skin-beautifying effect.

Japanese Unexamined Patent Publication No. 2014-19683 Japanese Unexamined Patent Publication No. 2009-40964

Biomolecules 2015,5,545-589

Increasing the expression of peroxiredoxin in cells has attracted attention not only in the field of cosmetology but also in the field of disease prevention and treatment.
Therefore, an object of the present invention is to provide a novel agent for increasing the expression of peroxiredoxin.

Another object of the present inventors is to provide a screening method for a peroxiredoxin expression-enhancing agent.

Since it is known that sebum, especially free fatty acids, increase in summer, we present what effect oleic acid, a type of free fatty acid, has on the expression of peroxiredoxin in the epidermis. I examined.
As a result, the presence of oleic acid increases the expression of GM-CSF (Granulocyte Macrophage colony-stimulating Factor) in epidermal cells, and the presence of GM-CSF increases the expression of peroxiredoxin in epidermal cells. It was found that the expression of was reduced.
They found that the jujube (Ziziphus jujuba) extract has the effect of increasing the expression of peroxiredoxin, and in particular, the effect of increasing the expression of peroxiredoxin in the presence of the free fatty acid oleic acid. rice field.
The present inventor has completed the present invention based on the above findings.

The present invention that solves the above problems is a peroxiredoxin expression enhancer containing an extract of a plant belonging to the genus Jujube (Rhamnaceae) of the family Buckthorn (Rhamnaceae) as an active ingredient.

The peroxiredoxin expression-enhancing agent of the present invention is preferably intended to be used for reducing the expression of peroxiredoxin in epidermal cells due to an increase in free fatty acids in the skin.

Examples of the free fatty acid include oleic acid, which is a main component of free fatty acid in the skin.

In a preferred embodiment of the invention, the plant is jujube (Ziziphus jujuba).

A preferred form of the peroxiredoxin expression-enhancing agent of the present invention is an external preparation or an oral preparation.

Preferred forms of the peroxiredoxin expression-enhancing agent of the present invention further include a melanin production inhibitor. Examples of the melanin production inhibitor include compounds selected from tranexamic acid and its derivatives.
Thereby, an excellent peroxiredoxin expression increasing effect can be obtained.

The present invention also provides a screening method characterized in that the active ingredient of a peroxiredoxin expression-enhancing agent is screened using the expression level of peroxiredoxin in the presence of a free fatty acid as an index.

The peroxiredoxin expression enhancer of the present invention is excellent in the action of increasing the expression of peroxiredoxin in epidermal cells, which is decreased in the presence of free fatty acids.
The peroxiredoxin expression-enhancing agent of the present invention prevents or ameliorate the deterioration of skin function and the conditions and diseases caused by the decrease in skin function during the season (spring to summer) when free fatty acids such as oleic acid increase in the skin. Excellent therapeutic effect.

It is a figure which shows the change of the expression level of peroxiredoxin in the human keratinocyte by the addition of the jujube extract (Taiso extract). It is a figure (photograph) which shows the change of the expression level of GM-CSF in human keratinocytes by the addition of oleic acid. The circular image corresponds to the nucleus of the cell, and the punctate relatively white part around it corresponds to GM-CSF. It is a figure which shows the change of the expression level of peroxiredoxin in human keratinocytes by the addition of GM-CSF. It is a figure which shows the change of the expression level of peroxiredoxin in human keratinocytes by the addition of oleic acid, and the change of the expression level of peroxiredoxin in human keratinocytes by the addition of jujube extract (Taiso extract) in the presence of oleic acid. .. It is a figure which shows the change of the expression level of peroxiredoxin in human keratinocytes by the addition of tripalmitin. It is a figure which shows the change of the expression level of peroxiredoxin in human keratinocytes by the addition of the mixture of jujube extract (taiso extract) and tranexamic acid.

The peroxiredoxin expression enhancer of the present invention contains an extract of a plant belonging to the family Buckthorn (Rhamnaceae) Jujube (Ziziphus) as an active ingredient.

As the plant, jujube (Ziziphus jujuba) can be preferably used. As the site of use of jujube, it is preferable to include at least one of fruit, pericarp, fruit spike, seed, and seed coat, and it is particularly preferable to include fruit. As the extraction solvent, a polar solvent such as water or alcohol can be preferably used. For example, the extract is preferably obtained by drying and finely pulverizing the fruit, if necessary, and then extracting with a solvent selected from hot water, ethanol, hydrous ethanol, butylene glycol, and hydrous butylene glycol. For example, an aqueous solution containing 10 to 90% by mass, preferably 30 to 60% by mass of ethanol or 1,3-butylene glycol can be mentioned. Dried fruits of jujube or related species of plants are also called taiso. As the extract of Taisou, for example, Taisou extract manufactured by Ichimaru Falcos can be used.

The peroxiredoxin expression-enhancing agent of the present invention contains the above-mentioned extract as an active ingredient.
The peroxiredoxin expression-enhancing agent of the present invention is preferably in the form of an external preparation or an oral preparation. Examples of the external preparation include forms such as cosmetics, quasi-drugs, and external preparations for skin. Moreover, those dosage forms are not particularly limited. Among them, the form of a cosmetic that can be continuously used is preferable because of its use for increasing the expression of peroxiredoxin, and above all, the form of a lotion, a beauty essence, a milky lotion, a cream, a gel, a sun care product, etc. is preferable. ..
In addition, as the oral preparation, supplements having a dosage form such as tablets, granules, and drinks, and foods and drinks are preferable.

In the case of an external preparation, the content of the extract, which is the active ingredient in the peroxiredoxin expression-enhancing agent, is preferably 0.0001 to 10% by mass, more preferably 0.001 to 5% based on the dry mass. It is mass%. In the case of an oral preparation, it is preferably 0.01 to 80% by mass, more preferably 0.1 to 50, based on the dry mass.

Peroxiredoxin is known to play an important role in cell repair because its antioxidant activity is known and it prevents denaturation and damage of proteins, lipids and nucleic acids. Peroxiredoxin is also known to be involved in cell proliferation and apoptosis, and has been suggested to be involved in the pathogenesis of liver cancer, skin cancer, diabetes, Alzheimer's disease, Parkinson's disease and the like.

Therefore, the peroxiredoxin expression-enhancing agent of the present invention can be used as an antioxidant. In addition, the peroxyredoxin expression enhancer of the present invention has skin symptoms or diseases such as cell activation, anti-aging, dermatitis, pigmentation, cancer such as liver cancer and skin cancer, diabetes, Alzheimer's disease, Parkinson's disease, and arteriosclerosis. It can be used as an agent for the prevention, improvement or treatment of.
In addition, the peroxiredoxin expression-enhancing agent of the present invention is particularly useful for preventing or improving the deterioration of skin condition due to active oxygen. Such deterioration of the skin condition includes phenomena such as pigmentation, fine unevenness of the skin, conspicuous pores, texture, wrinkles, sagging, blemishes, dullness, and dryness.

As shown in Examples described later, the peroxiredoxin expression-enhancing agent of the present invention is excellent in the action of increasing peroxiredoxin expression in epidermal cells in the presence of oleic acid. Therefore, the peroxiredoxin expression-enhancing agent of the present invention is preferably used for reducing the expression of peroxiredoxin in epidermal cells due to an increase in free fatty acids such as oleic acid in the skin.

That is, the peroxiredoxin expression-enhancing agent of the present invention is useful for improving the skin condition in which free fatty acids such as oleic acid are increased. Increases in free fatty acids, mainly oleic acid, are known to occur in the spring-summer season (J. Soc. Cosmet. Chem. Jpn. 34 (4) 365-373 (2000)). Therefore, it is effective that the peroxiredoxin expression-enhancing agent of the present invention is in the form of an external preparation for skin or cosmetics used from spring to summer.

From this point of view, it is also preferable that the peroxiredoxin expression-enhancing agent of the present invention is in the form of an external preparation for skin such as sunscreen cosmetics in combination with an ultraviolet protective agent. By adopting such a form, melanin production due to ultraviolet rays is suppressed by the effect of the ultraviolet protective agent, and skin symptoms such as pigmentation caused by an increase in free fatty acids, which is a factor different from ultraviolet rays, are suppressed. It is possible to comprehensively improve the deterioration of skin condition that tends to occur in the spring-summer season.

Examples of the above UV protection agents include paraaminobenzoic acid-based UV absorbers, anthranilic acid-based UV absorbers, salicylic acid-based UV absorbers, cinnamic acid-based UV absorbers, benzophenone-based UV absorbers, sugar-based UV absorbers, 2- Examples thereof include ultraviolet absorbers such as (2'-hydroxy-5'-t-octylphenyl) benzotriazole and 4-methoxy-4'-t-butyldibenzoylmethane.

From the above viewpoint, the peroxiredoxin expression-enhancing agent of the present invention is preferably in the form of an external preparation for skin such as a whitening cosmetic in combination with a melanin production inhibitor. With such a form, melanin production due to ultraviolet rays can be suppressed, and skin symptoms such as pigmentation caused by an increase in free fatty acids, which is a factor different from ultraviolet rays, can be suppressed, and spring to summer. It is possible to comprehensively improve the deterioration of skin condition that tends to occur in the season.
Further, as shown in Examples described later, it was confirmed that the combination of the extract as an active ingredient and a melanin production inhibitor enhances the peroxiredoxin expression action of the extract as an active ingredient. Therefore, by combining these, the excellent effect of the peroxiredoxin expression enhancer can be obtained.

Examples of the above-mentioned melanin production inhibitor include tranexamic acid, an alkylamide of tranexamic acid, an ester, a tranexamic acid derivative such as a multimer, an alkylresorcinol such as 4-butylresorcinol, albutin, ascorbic acid, ascorbic acid glucoside and 3-О−. Ascorbic acid derivatives such as ethyl ascorbic acid are preferable, and tranexamic acid and tranexamic acid derivatives are particularly preferable. They may also form salts.

When a melanin production inhibitor is contained in combination, the content mass thereof is 0.0001 to 5 times, preferably 0.001 to 2 times, more preferably 0.01 to 1 times the active ingredient (dry mass). It can be doubled.

The content of the melanin production inhibitor is preferably 0.01 to 30% by mass, more preferably 0.1 to 10% by mass in the case of an external preparation. In the case of an oral preparation, it is usually 0.01 to 80% by mass, preferably 0.1 to 50% by mass, and more preferably 1 to 30% by mass.

The peroxiredoxin expression-enhancing agent of the present invention preferably has a free fatty acid content of 5% by mass or less, preferably 1% by mass or less, and particularly preferably not contained.

In addition to the active ingredient, the peroxiredoxin expression-enhancing agent of the present invention can contain any ingredient usually used in cosmetics as long as the effect of the invention is not impaired. Such optional components include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, martitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4. -None surface activity of polyols such as hexylene glycol, 1,2-hexanediol, 1,2-octanediol, fatty acid sequels (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, triethanolamine ether alkylsulfate, etc. Agents, cationic surfactants such as stearyltrimethylammonium chloride, benzalconium chloride, laurylamine oxide, imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyroxy 2-sodium) Salts, etc.), betaine-based surfactants (alkylbetaine, amide betaine, sulfobetaine, etc.), amphoteric surfactants such as acylmethyltaurine, sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin Fatty acids (glycerin monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hardened castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoki monostearate, etc.) Ethylene sorbitan, etc.), POE sorbit fatty acid esters (POE-sorbit monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.) , POE alkyl ethers (POE2-octyldodecyl ether, etc.), POE alkylphenyl ethers (POE nonylphenyl ether, etc.), Pluronic type, POE / POP alkyl ethers (POE / POP2-decyltetradecyl ether, etc.), Te Tronics, POE castor oil / cured castor oil derivatives (POE castor oil, POE cured castor oil, etc.), sucrose fatty acid esters, nonionic surfactants such as alkyl glucosides, sodium pyrrolidone carboxylate, lactic acid, sodium lactate, etc. Moisturizing ingredients, may be surface treated, mica, talc, kaolin, synthetic mica, charcoal Powders such as calcium acid, magnesium carbonate, silicic anhydride (silica), aluminum oxide, barium sulfate, surface-treated, cobalt oxide, ultramarine, dark blue, zinc oxide inorganic pigments, surface-treated May be a composite pigment such as an iron oxide titanium dioxide sintered body, may be surface-treated, pearl agents such as mica titanium, fish phosphorus foil, bismuth oxychloride, may be raked Red No. 202 , Red 228, Red 226, Yellow 4, Blue 404, Yellow 5, Red 505, Red 230, Red 223, Orange 201, Red 213, Yellow 204, Yellow 203, Blue Organic pigments such as No. 1, Green No. 201, Purple No. 201, Red No. 204, Organic powders such as polyethylene powder, polymethylmethacrylate, nylon powder, organopolysiloxane elastomer, lower alcohols such as ethanol and isopropanol. , vitamin a or its derivatives, vitamin _{B 6} hydrochloride, vitamin _{B 6} tripalmitate, vitamin _{B 6} dioctanoate, vitamin _{B 2} or derivatives thereof, vitamin _{B 12,} vitamin B such as vitamin _{B 15} or a derivative thereof, alpha- Examples thereof include vitamin Es such as tocopherol, β-tocopherol, γ-tocopherol and vitamin E acetate, and vitamins such as vitamin Ds, vitamin H, pantothenic acid, pantetin and pyroquinolinquinone.

The screening method of the present invention is characterized in that the active ingredient of a peroxiredoxin expression-enhancing agent is screened using the expression level of peroxiredoxin in the presence of a free fatty acid as an index.

As shown in the examples below, it was found that the expression of peroxiredoxin was reduced in the presence of free fatty acids such as oleic acid. On the other hand, as described above, it is known that free fatty acids such as oleic acid in the skin increase from spring to summer. Based on these findings, if a component that increases the expression of peroxiredoxin in the presence of free fatty acids can be screened, it will be possible to improve the skin condition from spring to summer from the viewpoints of conventional UV protection, suppression of melanin production, etc. It is possible to screen useful components in terms of increased expression of antioxidant enzymes in the biological skin, which is different from the above.

The screening method of the present invention includes adding a free fatty acid such as oleic acid and a test substance to a culture system of epidermal cells, and measuring the expression level of peroxiredoxin. The expression level of peroxiredoxin can be measured by a conventional method such as immunoassay or qRT-PCR.

In addition, by carrying out the screening method in the presence of free fatty acids such as oleic acid, it becomes possible to screen components that increase the expression of peroxiredoxin in the presence of free fatty acids.

<Test Example 1> Jujube extract addition test This test was conducted for the purpose of measuring the change in the expression level of peroxiredoxin due to the addition of jujube extract.
In this test, Ichimaru Falcos' Taisou extract was used as the jujube extract (the same applies to the following test examples). As a comparative example, a rosehip extract manufactured by Maruzen Pharmaceuticals Co., Ltd. was used.

Newborn keratinocytes (manufactured by Kurabo Industries Ltd.) were ^{sown at 5.0 × 10 4} cell / well and cultured for 24 hours. Subsequently, 1% by mass of each of Taisou extract and Rosehip extract was added, and the cells were cultured for 24 hours. After completion of the culture, cells were collected and the mRNA expression level of peroxiredoxin was measured by qRT-PCR. As a control, the one obtained by adding only the solvent and culturing in the same manner was used.

The results are shown in FIG.
As shown in FIG. 1, when the taiso extract was added, the expression level of peroxiredoxin was increased with respect to the control, whereas when the rosehip extract was added, the expression level of peroxiredoxin was increased. It was confirmed that it could not be seen.
From this, it was clarified that the jujube extract has an action of increasing the expression of peroxiredoxin. In addition, it was clarified that the rosehip extract, which is known to have an antioxidant effect, does not have an effect of increasing the expression of peroxiredoxin.

<Test Example 2> Jujube extract addition test in the presence of free fatty acids This test was conducted for the purpose of measuring GM-CSF expression, peroxiredoxin expression, and the action of jujube extract on them due to the addition of free fatty acids.

(1) GM-CSF expression by addition of oleic acid This test was conducted for the purpose of measuring the change in the expression level of GM-CSF by the addition of free fatty acid. In this test, oleic acid (manufactured by Wako Pure Chemical Industries, Ltd.), which is one of the major free fatty acids in sebum, was used as the free fatty acid.

Newborn keratinocytes (manufactured by Kurabo Industries Ltd.) were ^{sown at 7.0 × 10 4} cell / well and cultured for 24 hours. Subsequently, 5 μM of oleic acid was added, and the cells were cultured for 24 hours. After completion of the culture, the cells were fixed with 4% paraformaldehyde, immunostained with an anti-GM-CSF antibody (manufactured by Abcam Co., Ltd.), and observed under a microscope. As a control, those cultured in the same manner without adding oleic acid were used.

The results are shown in FIG.
As shown in FIG. 2, in the sample to which oleic acid was added, an increase in the expression level of GM-CSF in the cells was observed as compared with the control in which this was not added (an increase in red fluorescence was detected around the nucleus). ..
From this, it was shown that the expression of GM-CSF was increased by the presence of free fatty acids such as oleic acid.

(2) Expression of peroxiredoxin by addition of GM-CSF This test was conducted for the purpose of measuring the effect of GM-CSF on the expression of peroxiredoxin.
Newborn keratinocytes (manufactured by Kurabo Industries Ltd.) were ^{sown at 5.0 × 10 4} cell / well and cultured for 24 hours. Subsequently, 50 ng / mL of GM-CSF was added and cultured for 24 hours. After completion of the culture, cells were collected and the mRNA expression level of peroxiredoxin was measured by qRT-PCR. As a control, the one obtained by adding only the solvent and culturing in the same manner was used.

The results are shown in FIG.
As shown in FIG. 3, in the sample to which GM-CSF was added, a decrease in the expression level of peroxiredoxin in the cells was observed as compared with the control to which this was not added.
This indicates that the presence of GM-CSF reduces the expression of peroxiredoxin.

Considering the results of (1) and (2), it can be seen that the addition of oleic acid increases the expression level of GM-CSF in cells, which in turn decreases the expression level of peroxiredoxin in cells.

(3) Jujube extract addition test The purpose of this test is to measure the change in peroxiredoxin expression level due to the addition of free fatty acid, which is a component of sebum, and the change in peroxiredoxin expression level due to the addition of jujube extract. I went there.
In this test, oleic acid, which is one of the major free fatty acids in sebum, was used.

Newborn keratinocytes (manufactured by Kurabo Industries Ltd.) were ^{sown at 5.0 × 10 4} cell / well and cultured for 24 hours. Subsequently, 5 μM of oleic acid or 5 μM of oleic acid and 1% by mass of Taisou extract were added and cultured for 24 hours. After completion of the culture, cells were collected and the mRNA expression level of peroxiredoxin was measured by qRT-PCR. As a control, the one obtained by adding only the solvent and culturing in the same manner was used.

The results are shown in FIG.
As shown in FIG. 4, it was confirmed that the expression level of peroxiredoxin was reduced by adding oleic acid. It was also confirmed that the addition of oleic acid and taiso extract increased the expression level of peroxiredoxin to the same level as the control for the system to which oleic acid was added.
From this, it was clarified that the jujube extract has an action of increasing the expression of peroxiredoxin. In particular, it was revealed that the jujube extract has an action of increasing the expression of peroxiredoxin in the presence of free fatty acids such as oleic acid.

(4) Triglyceride addition test In this test, changes in the expression level of peroxiredoxin due to the addition of triglyceride, which is a component of sebum, were confirmed, and the decrease in the expression level of peroxiredoxin was peculiar to the presence of free fatty acids. I went there to make sure it was there.
In this study, tripalmitin was used as the triglyceride.

Newborn keratinocytes (manufactured by Kurabo Industries Ltd.) were ^{sown at 5.0 × 10 4} cell / well and cultured for 24 hours. Subsequently, 10 μM of tripalmitin was added and cultured for 24 hours. After completion of the culture, cells were collected and the mRNA expression level of peroxiredoxin was measured by qRT-PCR. As a control, the one obtained by adding only the solvent and culturing in the same manner was used.

The results are shown in FIG.
As shown in FIG. 5, in the sample to which tripalmitin was added, a decrease in the expression level of peroxiredoxin in the cells was not observed as compared with the control to which this was not added.

From the results of (3) and (4), it was shown that the decrease in the expression of peroxiredoxin is specifically caused by the presence of free fatty acid, which is one of the sebum components.

<Test Example 3> Test for adding mixture of nut extract and melanin production inhibitor This test was conducted for the purpose of measuring the change in the expression level of peroxiredoxin due to the addition of a mixture of nut extract and melanin production inhibitor.
Tranexamic acid was used as a melanin production inhibitor.

Newborn keratinocytes (manufactured by Kurabo Industries Ltd.) were ^{sown at 5.0 × 10 4} cell / well and cultured for 24 hours. Subsequently, 1% by mass of Taiso extract, 1% by mass of Taiso extract and 400 μg / mL of tranexamic acid were added, and 400 μg / mL of tranexamic acid were added and cultured for 24 hours. After completion of the culture, cells were collected and the mRNA expression level of peroxiredoxin was measured by qRT-PCR. As a control, the one obtained by adding only the solvent and culturing in the same manner was used.

The results are shown in FIG.
As shown in FIG. 6, it was confirmed that when a mixture of taiso extract and tranexamic acid was added, the expression level of peroxiredoxin was significantly increased as compared with the case where tranexamic acid was added alone. On the other hand, it was confirmed that when tranexamic acid was added alone, the expression level of peroxiredoxin was not increased.
From this, it was clarified that the combination of the jujube extract and tranexamic acid enhances the action of increasing the expression of peroxiredoxin in the jujube extract.

<Example 1> Cosmetics Toners, which are the cosmetics of the present invention, were prepared according to the formulations shown in Table 1 below.

<Example 2> Cosmetics Toners, which are the cosmetics of the present invention, were prepared according to the formulations shown in Table 2 below.

<Example 3> Foods The foods of the present invention were prepared according to the formulations shown in Table 3 below. That is, the prescription ingredient was granulated in a rolling phase (“Numal Melizer” manufactured by Fuji Powder Co., Ltd.) together with 10 parts by weight of water, and tableted to obtain 1 g of tablet-shaped food.

The present invention can be applied to cosmetics, supplements, foods and drinks.

Claims (3)

A screening method comprising screening an active ingredient of a peroxiredoxin expression-enhancing agent using the expression level of peroxiredoxin in the presence of a free fatty acid as an index. The screening method according to claim 1, wherein the expression level of peroxiredoxin is measured by adding a free fatty acid and a test substance to a culture system of epidermal cells. The screening method according to claim 1 or 2, wherein the free fatty acid is oleic acid.

Images